Is developmental plasticity triggered by DNA methylation changes in the invasive cane toad (Rhinella marina)?

Abstract Many organisms can adjust their development according to environmental conditions, including the presence of conspecifics. Although this developmental plasticity is common in amphibians, its underlying molecular mechanisms remain largely unknown. Exposure during development to either ‘cannibal cues’ from older conspecifics, or ‘alarm cues’ from injured conspecifics, causes reduced growth and survival in cane toad (Rhinella marina) tadpoles. Epigenetic modifications, such as changes in DNA methylation patterns, are a plausible mechanism underlying these developmental plastic responses. Here we tested this hypothesis, and asked whether cannibal cues and alarm cues trigger the same DNA methylation changes in developing cane toads. We found that exposure to both cannibal cues and alarm cues was associated with local changes in DNA methylation patterns. These DNA methylation changes affected genes putatively involved in developmental processes, but in different genomic regions for different conspecific‐derived cues. Genetic background explains most of the epigenetic variation among individuals. Overall, the molecular mechanisms triggered by exposure to cannibal cues seem to differ from those triggered by alarm cues. Studies linking epigenetic modifications to transcriptional activity are needed to clarify the proximate mechanisms that regulate developmental plasticity in cane toads.


| INTRODUC TI ON
Developmental plasticity is the ability of a single genotype to give rise to a range of phenotypes in different environments.Plasticity can be adaptive when environmental conditions are predictable and can involve both short-and long-term changes in physiology, morphology, life-history traits and behaviour (Sultan, 2003;West-Eberhard, 2003).Although studies on the underlying molecular mechanisms of plasticity are burgeoning, these mechanisms remain poorly understood (Gilbert & Epel, 2015;Lafuente & Beldade, 2019;Sommer, 2020).
Amphibians are good models to study molecular mechanisms of phenotypic plasticity because the growth and development of their aquatic larvae are susceptible to environmental factors such as food availability, predator exposure, conspecific density, and pond drying (Denver, 2021;Newman, 1992;Wilbur & Collins, 1973).The invasive cane toad, Rhinella marina, is a good exemplar of a species capable of altering its developmental trajectory in response to environmental conditions (Lever, 2001).
Female cane toads lay large clutches (up to several tens of thousands of eggs) asynchronously in ponds, resulting in overlapping cohorts of developing offspring where egg and larvae densities can reach extreme levels (Alford et al., 1995;DeVore, Crossland, & Shine, 2021).Cannibalism of eggs and hatchlings by older tadpoles is common in these breeding ponds, such that survival of newlylaid eggs to the free-swimming tadpole stage is often <1% (Alford et al., 1995;DeVore, Crossland, & Shine, 2021).
Both alarm cues and cannibal cues thus induce developmental plastic responses and reduce growth and survival in cane toad tadpoles.Moreover, both cues likely have long-term detrimental effects post-metamorphosis, affecting adult survival against cannibalism (Pizzatto & Shine, 2008), predation (Ward-Fear et al., 2012), parasitism (Kelehear et al., 2009) and desiccation (Child et al., 2008).Finally, these cues appear mechanistically associated with one another, because exposure to alarm cues can have intergenerational effects by increasing the potency of cannibal cues in the next generation (Sarma et al., 2021).
In this study, we asked whether exposure to cannibal cues and exposure to alarm cues might trigger the same molecular mechanisms.Specifically, we tested (i) whether exposure to conspecific cues triggers changes in DNA methylation patterns in cane toad tadpoles that might then induce developmental plasticity, and (ii) whether both cannibal cues and alarm cues trigger the same epigenetic modifications.We focused on DNA methylation, because this epigenetic mechanism can influence transcriptional activity on the one hand (Jaenisch & Bird, 2003), and can be affected by environmental factors on the other (Dowen et al., 2012;Radford et al., 2014), and because studies of cane toads have shown that exposure to alarm cues changes DNA methylation patterns in tadpoles (Sarma et al., 2020(Sarma et al., , 2021)).Changes in DNA methylation are thus a plausible molecular mechanism that might underlie developmental plasticity in cane toads.

| Toads
We collected adult cane toads from two genetically distinct populations (Selechnik et al., 2019) within the Australian invasive range: spawning (see DeVore, Crossland, and Shine (2021) for details).
We then kept eggs in aerated holding tanks for 72 h to ensure successful fertilisation.

| Experimental design
In this study, we compared the effect of conspecific exposure on DNA methylation during development in two contexts: exposure to cannibal cues and exposure to alarm cues.

| Cannibal cue experiment
In this experiment, we exposed focal hatchlings from eight clutches (i.e., 4 range-core and 4 range-edge clutches) to three treatments: (1) exposure to conspecific tadpoles (i.e., cannibal cues) from clutch i, (2) exposure to conspecific tadpoles from clutch j, (3) control (no conspecific tadpoles added).Clutches i and j were raised from clutches from the same locality as the focal hatchlings but were included so that we could measure the impact of genotype on the effect of cannibal cues.We used 1 L-treatment tanks filled with 750 mL of water from a local aquifer.In the cannibal cue treatments, we first added three captive-raised tadpoles [developmental stage 30-35 (Gosner, 1960)] to each tank, separated by a 1 × 1 mm fly screen mesh from the compartment where focal hatchlings were placed.This allowed chemical cannibal cues to diffuse throughout the container but prevented cannibalism (Crossland & Shine, 2012).Several hours after conspecific tadpoles were added, we randomly allocated five hatchlings [developmental stage 16/17, i.e., approximately 1 day post-hatching (Gosner, 1960)] to each treatment tank.We replicated each treatment three times.We removed the conspecific tadpoles after 24 h and left the developing hatchlings for a further 24 h, by which time they had developed into free-swimming, feeding tadpoles (stage 25).We then transferred the stage 25 tadpoles to new tanks and fed them blended Hikari algae wafers (Kyorin, Japan) ad libitum, with fresh water changes every 3 days.Ten days later, we humanely euthanised all tadpoles (stage ~32) and three tadpoles from each tank were blotted, weighed, and then frozen prior to DNA extraction.

| Alarm cue experiment
This experiment was described in Sarma et al. (2020).Briefly, we randomly allocated two hatchlings from each clutch to two treatments: (1) exposure to conspecific alarm cues (n = 11 clutches, each region), ( 2) control (n = 10 clutches, range core; n = 9 clutches, range edge).We used hatchlings from the same clutches for each of these two treatments, unless mortality prevented us from doing so (i.e., three cases where only one hatchling was available and was allocated to the alarm cue treatment).In the alarm cue treatment, we added to each tank 4 mL of water containing the freshly-macerated bodies of two conspecific tadpoles (Hagman et al., 2009)

| Reduced-representation bisulfite sequencing
We extracted DNA from whole tadpoles using a Gentra Puregene Kit (Qiagen) according to the manufacturer's instructions.We prepared reduced-representation bisulfite sequencing (RRBS) libraries

| DNA methylation analyses
We used FastQC 0.11.8 (Andrews, 2010) to assess the quality of the reads.We trimmed adapter sequences and low-quality reads using Trim Galore 6.5 (Krueger, 2015).We then mapped the remaining reads to the cane toad genome (Edwards et al., 2018) using Bismark 0.22.3(Krueger & Andrews, 2011) with HISAT2 2.1.0(Kim et al., 2019).We extracted methylation status for each CpG using Bismark.We carried out downstream differential methylation analyses using the package methylKit (Akalin et al., 2012) in R 4.0.4(R Core Team, 2021).We merged strands for each CpG, and we only kept sites that had a depth of coverage of at least 10 reads for subsequent analyses.We further filtered out any site with a coverage higher than the 99.9th percentile of read counts.We deemed CpGs as methylated (hereafter, mCpGs) when their methylation level (i.e., the ratio of C to [C + T] reads at each CpG) was >10%.We defined differentially methylated cytosines (hereafter, DMCs) as CpGs with a methylation difference of 20% or greater between the two groups (i.e., conspecific-cue exposed vs controls), a q-value (Fisher's exact test corrected p-value) (Wang et al., 2011) of 0.05 or less, and that were present in at least three samples in each group.We used the R package eDMR (Li et al., 2013) to identify differentially methylated regions (hereafter, DMRs).DMRs were defined as regions with a mean methylation difference of at least 20% between the two groups, a q-value of 0.05 or less, and that contained at least 5 CpGs and 3 DMCs.

| Effect of conspecific cues on tadpole growth
For each experiment, we investigated whether the exposure to conspecific cues influenced mass of focal tadpoles, using linear mixed-effects models (LMMs) with the R package lme4 (Bates et al., 2015).We included mass as the dependent variable, treatment as a fixed effect, and clutch ID and replicate as random factors.

| RE SULTS
The average mass of tadpoles that had been exposed to cannibal cues as hatchlings was lower than controls in range-edge populations, but not in range-core populations (LMMs, respectively p < .00001and p = .129;Figure 1b,c).Alarm cues had no significant effect on mean mass of exposed tadpoles in either range-core or range-edge populations (both p > .396; Figure 1d,e).
After filtering and quality trimming, the breadth of coverage (i.e., the number of CpGs with a coverage ≥10×) was 2.6 ± 0.1 million CpGs (mean ± SE) for tadpoles from the cannibal cue experiment, and 2.5 ± 0.1 million CpGs for tadpoles from the alarm cue experiment (Table A1 in Appendix).The depth of coverage was respectively 16.6 ± 0.1 and 16.8 ± 0.2 fold (Table A1 in Appendix).
The methylation density (i.e., the ratio of mCpGs to CpGs across the covered genome) was very high across all samples, as typically observed in vertebrates (Bird, 2002).Nonetheless, tadpoles exposed to either cannibal cues or alarm cues had higher methylation densities compared to controls in both range-core and range-edge populations (generalised linear mixed-effects models [GLMMs], all p < .00001; Figure 2a-d).Likewise, the proportion of fully methylated sites was higher in tadpoles exposed to either cannibal cues or alarm cues compared to controls in both range-core and range-edge populations (GLMMs, all p < .00001; Figure 2e-h).Finally, the methylation level was also higher in tadpoles exposed to either cannibal cues or alarm cues compared to controls in both range-core and range-edge populations (GLMMs, all p < .00001; Figure 2i-l).
Hierarchical clustering based on methylation levels revealed a clear clustering by clutch identity for both range-core and rangeedge populations (Figure 3).This pattern indicates that genetic differences were the main driver of epigenetic differences between samples, whereas conspecific cue exposure had a comparatively minor (albeit statistically significant) effect.
Across all groups, the number of DMCs in cue-exposed tadpoles compared to controls ranged from 8131 to 13,582, of which 54.1%-61.4% were hypermethylated in tadpoles exposed to either cannibal cues or alarm cues (Figure 4a-d).Most DMCs (88.8% and 89.3% of hypermethylated and hypomethylated DMCs, respectively) were specific to tadpoles from one population exposed to one treatment (Figure 4e,f).Only 2 DMCs had higher methylation levels across all cue-exposed tadpoles compared to controls, none of them intersecting with gene bodies (Figure 4e).No hypomethylated DMC was common across all groups (Figure 4f).
There were 34 DMRs in range-core tadpoles exposed to cannibal cues compared to controls, of which 20 (58.8%) were located within genes (Figure 5a and Table A2 in Appendix).In range-edge tadpoles exposed to cannibal cues, there were 74 DMRs compared to controls, out of which 30 (40.5%) were found within genes, and 4 (5.4%) were located in promoter regions (Figure 5b and Table A3 in Appendix).DMRs each contained on average 5.4 ± 2.9 DMCs (mean ± SD; range 3-13) and 5.9 ± 3.0 DMCs (range 3-17) in rangecore and range-edge tadpoles, respectively.The majority of DMRs were hypermethylated in cannibal-cue-exposed tadpoles compared to controls in both populations (respectively 64.7% and 60.8%; Further, only two overlapping DMRs (intersecting with FAM168A and MAPK14) showed parallel changes in DNA methylation levels in both populations, while the other four showed opposite changes in range-core and range-edge tadpoles compared to controls.
There were 32 DMRs between range-core tadpoles exposed to alarm cues and controls, out of which 14 (43.8%) were found within genic regions, and 3 (9.4%)were found in promoter regions (Figure 5c and Table A4 in Appendix exposed to alarm cues, there were 53 DMRs compared to controls, out of which 27 (50.9%)were located within genes, and 1 (1.9%) was found in promoter regions (Figure 5d and Table A5 in  and A5 in Appendix).
Only 1 DMR, located within the GUCA1A gene, overlapped and showed an increase in DNA methylation levels across both populations in tadpoles exposed to alarm cues compared to controls (Figure 5e).
Overall, there was minimal overlap in regions showing differential methylation in tadpoles exposed to cannibal cues and in tadpoles exposed to alarm cues compared to controls (Figure 5e).
Only 2 DMRs overlapped between cannibal-cue-exposed and alarm-cue-exposed tadpoles from range-core populations.These There was no DMR overlap across both treatments and both populations (Figure 5e).GO enrichment analyses did not reveal any significantly over-represented GO term for all DMR lists.Our findings reveal that exposure to conspecific cannibal cues and alarm cues were both associated with changes in the DNA methylation profiles of cane toad tadpoles, but in different ways.
Our results thus suggest that the developmental plastic responses seen in these two contexts, despite their similar short-and longterm consequences, are underpinned by distinct molecular mechanisms.
We did observe a similar overall pattern of hypermethylation in tadpoles exposed to both conspecific cues in both populations compared to controls.These slightly higher levels of DNA methylation might indicate marginally lower levels of gene expression (Jaenisch & Bird, 2003) in tadpoles exposed to conspecific cues.Nonetheless, these changes were small (typically <1%), and the relationship between DNA methylation levels and transcriptional activity are far from being universal and unidirectional (de Mendoza et al., 2020).
We also observed some overlap in DMRs between cannibalcue-exposed tadpoles compared to controls and alarm-cueexposed tadpoles compared to controls.Two DMRs overlapped between both treatments in range-core tadpoles, and 9 DMRs overlapped between both treatments in range-edge tadpoles.
These DMRs were found within the gene bodies of AKTIP-A, HSPB6, PGS1, SCNN1G, SOCS3, as well as two uncharacterised genes.AKTIP-A mouse mutants show developmental abnormalities (Anselme et al., 2007).HSPB6 is involved in the regulation of angiogenesis (Vafiadaki et al., 2020), SCNN1G plays a role in water homeostasis (Hobbs et al., 2013), while SOCS3 is involved in the regulation of food intake (Zhu et al., 2021).DNA methylation patterns of 5′-flanking regions have been shown to correlate with gene expression, at least for SCNN1G (Pierandrei et al., 2021).
Changes in DNA methylation in these genes were already evidenced in cane toad tadpoles following exposure to alarm cues (Sarma et al., 2020(Sarma et al., , 2021)), and we here show that the same modifications occur after exposure to cannibal cues.These findings offer a possible mechanistic link explaining why individuals exposed to alarm cues produce offspring that emit more potent cannibal cues (Sarma et al., 2021).
Changes in DNA methylation levels in the above-mentioned and SOX5 play a role in brain development (Andrews et al., 2016;Palmer et al., 2016;Stolt et al., 2006).LARGE1, MAPK14, NLK, RANBP3L and RYK are involved in skeletal system development (Chen et al., 2015;Goddeeris et al., 2013;Greenblatt et al., 2010;Halford et al., 2000;Zanotti & Canalis, 2012).LRP4 plays a role in normal organ development (Weatherbee et al., 2006).NIN is integral to epidermis development (Lecland et al., 2019).SEZ6 is involved in the regulation of motor functions (Gunnersen et al., 2020).Alarm-cue-exposed tadpoles showed differences in DNA methylation levels in the genes CEP135, DACH2, ECE1, EIF3A, ENG, GPR155, MINK1, MYL3, PKD1, TIE1 and ZCCHC3.CEP135 and MINK1 are involved in brain development (Bamborschke et al., 2020;Dan et al., 2000).DACH2 plays a role in muscle development (Tang & Goldman, 2006).ECE1, ENG, MYL3 and PKD1 are involved in heart development (Arthur et al., 2000;Boulter et al., 2001;James et al., 1999;Poltavski et al., 2019).EIF3A plays a role in intestinal development (Liu et al., 2007).TIE1 is involved in angiogenesis (Loughna & Sato, 2001).ZCCHC3 plays a role in innate immune response (Lian et al., 2018).Finally, GPR155 is involved in cognitive functions (Nishimura et al., 2007).Overall, these genes are interesting candidates for future studies, whose main focus should be directed towards generating gene expression data for tadpoles exposed to both conspecific cues and controls across development.This should help to confirm that the above-mentioned genes that show differences in DNA methylation levels between treatments also show differences in gene expression levels, and should bring us one step closer to establishing a causal link between molecular mechanisms and developmental plasticity.
Our results revealed clearly that epigenetic differences between tadpoles were mostly driven by their clutch identity.This phenomenon has previously been documented in cane toads (Sarma et al., 2020), and indicates that genetic differences between individuals are the main cause for their divergence in DNA methylation patterns.The influence of genotypic variation on DNA methylation marks appears ubiquitous.Mounting evidence shows that, although epigenetic marks can be modified by environmental exposure, in many cases they do so under genetic control (Do et al., 2017;Gaunt et al., 2016;Hannon et al., 2018;Kerkel et al., 2008;Min et al., 2021;Tycko, 2010;Villicaña & Bell, 2021).These results stress the importance of controlling for genetic effects (i.e., having a balanced experimental design in terms of clutch identity) when investigating differences in DNA methylation patterns between treatment and control.These results also help explain why we observed only minimal overlap in DMRs between range-core and range-edge tadpoles, even within each treatment (i.e., for tadpoles exposed to the same conspecific cue).Because tadpoles from distinct populations necessarily came from distinct clutches, their constitutive genetic differences induced population-specific DNA methylation patterns (prior to any conspecific cue exposure) that were of greater magnitude than the effect of treatment itself.Our findings further complement previous results showing that clutches vary in their reaction norms, i.e., in their propensity to accelerate their development, when exposed to conspecific cues (DeVore, Crossland, & Shine, 2021).
We found that changes in DNA methylation following exposure to both cannibal cues and alarm cues were largely population-specific, and were of greater magnitude in range-edge tadpoles than in rangecore tadpoles.This effect was mirrored in the developmental effects of conspecific cue exposure (i.e., greater effect on mass at the rangeedge).These population-specific DNA methylation patterns are consistent with previous studies investigating epigenetic patterns in cane toads (Sarma et al., 2020(Sarma et al., , 2021)).More generally, they are consistent with the well documented between-population differences in cane toad morphology (Hudson et al., 2016;Phillips et al., 2006), physiology (Brown et al., 2015), behaviour (Gruber et al., 2017;Lindstrom et al., 2013), transcriptomics (Rollins et al., 2015;Yagound, West, Richardson, Gruber, et al., 2022;Yagound, West, Richardson, Selechnik, et al., 2022) and genetics (Selechnik et al., 2019) across the Australian invasive range.
We did not detect any significant reduction in body mass in alarm-cue-exposed tadpoles.By contrast, previous studies found such an effect at a later stage in development (i.e., at metamorphosis) (Crossland et al., 2019;Hagman et al., 2009;Hagman & Shine, 2009).
Thus, the apparent lack of growth reduction seen following alarmcue exposure may be an artefact of early euthanasia.

TA B L E A 2
DMRs between range-core tadpoles exposed to cannibal cues and controls.

ADAMTS14
A disintegrin and metalloproteinase with thrombospondin motifs 14 a Also found between range-core tadpoles exposed to alarm cues and controls.
b Also found between range-edge tadpoles exposed to alarm cues and controls.
c Also found between range-edge tadpoles exposed to cannibal cues and controls.
TA B L E A 3 DMRs between range-edge tadpoles exposed to cannibal cues and controls.b Also found between range-edge tadpoles exposed to alarm cues and controls.

Contig
c Also found between range-core tadpoles exposed to cannibal cues and controls.

TA B L E A 3 (Continued)
TA B L E A 4 DMRs between range-core tadpoles exposed to alarm cues and controls.Also found between range-edge tadpoles exposed to alarm cues and controls.
b Also found between range-core tadpoles exposed to cannibal cues and controls.
c Also found between range-edge tadpoles exposed to cannibal cues and controls.
TA B L E A 5 DMRs between range-edge tadpoles exposed to alarm cues and controls.b Also found between range-core tadpoles exposed to cannibal cues and controls.

Contig
c Also found between range-edge tadpoles exposed to cannibal cues and controls.
TA B L E A 5 (Continued) on each of 10 consecutive days (days 7-16 post spawning).In the control treatment, we added 4 mL of non-chlorinated water on 10 consecutive F I G U R E 1 Populations used and effect of exposure to conspecific cues on tadpole growth.(a) Location of samples.Samples used in the cannibal cue experiment originated from localities depicted by circles.Samples used in the alarm cue experiment originated from localities depicted by triangles.Orange and green localities respectively correspond to range-core and range-edge populations.NT, Northern Territory; QLD, Queensland; WA, Western Australia.The shaded area represents the cane toad's Australian invasive range.(b, c) Mass (g) of tadpoles exposed to cannibal cues and controls from (b) range-core and (c) range-edge populations, and of tadpoles exposed to alarm cues and controls from (d) range-core and (e) range-edge populations.Violin plots represent median, interquartile range (IQR), 1.5 × IQR, and kernel density plot.Significant p-values (LMMs) are highlighted in bold.

Figure
Figure 5a,b and Tables A2 and A3 in Appendix).Only 6 DMRs overlapped across both populations in tadpoles exposed to cannibal cues compared to controls (Figure5e), out of which four were located within genes FAM168A, RYK, MAPK14, and HYDIN.
).In range-edge tadpoles F I G U R E 3 Hierarchical clustering (average agglomerative method on correlation distances) of samples according to their DNA methylation status.(a) All samples.(b, c) Samples exposed to cannibal cues and controls from (b) range-core and (c) range-edge populations.(d, e) Samples exposed to alarm cues and controls from (d) range-core and (e) range-edge populations.Coloured boxes group samples by clutch, treatment (i.e., red, alarm cue; dark green, cannibal cue; grey, control) and population (i.e., orange, range-core; light green, range-edge). 0 Appendix).Each DMR contained on average 4.6 ± 2.1 DMCs (range 3-12) in range-core tadpoles, and 6.6 ± 4.2 DMCs (range 3-25) in range-edge tadpoles.As for cannibal-cue-exposed tadpoles, most DMRs showed a significant increase in DNA methylation level compared to controls in both range-core and range-edge populations (respectively 78.1% and 66.0%; Figure5c,dand TablesA4 DMRs were located respectively in the AKTIP-A gene, and in an intergenic region.The first DMR showed opposite changes in DNA methylation levels in both treatments relative to controls, while the second one showed hypermethylation in both treatments relative to controls.In range-edge tadpoles, 9 DMRs overlapped between tadpoles exposed to cannibal cues and those exposed to alarm cues.Four of those DMRs intersected with genes HSPB6, PGS1, SCNN1G, SOCS3, as well as the uncharacterised transcript RMA_00054127.However, only genes PGS1, SCNN1G and SOCS3 exhibited parallel changes in DNA methylation levels in both treatments relative to controls (hypomethylation for PGS1 and SOCS3 and hypermethylation for SCNN1G).Of the 5 remaining intergenic DMRs, 3 showed parallel changes and 2 showed opposite changes in DNA methylation levels in both treatments relative to controls.One DMR (showing opposite changes in DNA methylation levels and intersecting with uncharacterised transcript RMA_00000723) overlapped between range-core tadpoles exposed to cannibal cues and range-edge tadpoles exposed to alarm cues.Lastly, three DMRs overlapped between range-edge tadpoles exposed to cannibal cues and range-core tadpoles exposed to alarm cues.Only one of those DMRs was located within gene PKP4 and showed opposite changes in DNA methylation levels in both treatments relative to controls.

F
I G U R E 4 DNA methylation changes in tadpoles exposed to cannibal cues and alarm cues.(a-d) Volcano plots of DMCs between tadpoles exposed to cannibal cues and controls from (a) range-core and (b) range-edge populations, and between tadpoles exposed to alarm cues and controls from (c) range-core and (d) range-edge populations.Nonsignificant sites are represented in grey.(e, f) Venn diagrams represent the overlap of (e) hypermethylated and (f) hypomethylated DMCs in cannibal-cue-exposed tadpoles versus controls and in alarmcue-exposed tadpoles versus controls, both from range-core and range-edge populations.
hypermethylated in cue-exposed tadpoles vs controls DMCs hypomethylated in cue-exposed tadpoles vs controls genes may have developmental consequences during cane toads' larval life.Yet, as well as being restricted to only a few overlapping regions, changes in DNA methylation levels were not always consistent across treatments (e.g., hypermethylation in one case and hypomethylation in the other).This casts doubts on the causative F I G U R E 5 Local DNA methylation changes in tadpoles exposed to cannibal cues and alarm cues.(a-d) Volcano plots of DMRs between tadpoles exposed to cannibal cues and controls from (a) range-core and (b) range-edge populations, and between tadpoles exposed to alarm cues and controls from (c) range-core and (d) range-edge populations.Nonsignificant regions are represented in grey.(e) Venn diagram representing the overlap of DMRs in cannibal-cue-exposed tadpoles versus controls and in alarm-cue-exposed tadpoles versus controls, both from range-core and range-edge populations.Genes intersecting with overlapping DMRs are indicated.in DNA methylation levels, changes in gene expression levels, and downstream phenotypic consequences due to exposure to cannibal cues and alarm cues.The overlap in DMCs was also minimal, and it is unclear mechanistically how changes in DNA methylation levels of single CpGs can affect gene activity (but see e.g., Sobiak & Leśniak, 2019).Within each treatment, additional DMRs intersected with genes also putatively involved in developmental processes.For example, cannibal-cue-exposed tadpoles showed differences in DNA methylation levels in the genes HDYN, LARGE1, LRP4, MAPK14,NIN, NLK, PLXNA1, RANBP3L, RYK, SEZ6 and SOX5.HDYN, PLXNA1 Also found between range-core tadpoles exposed to alarm cues and controls.

a
Also found between range-core tadpoles exposed to alarm cues and controls.
By contrast, it is also possible that these gene networks are quite similar between both contexts, but that we were only able to capture a fraction of the genomic regions involved in each case.The lack of overlap could then derive from constraints in DNA methylation statistics for each sample.